SPR is the resonant excitation of oscillating free charges at the interface of a metal and a dielectric. When SPR spectra are generated and collected, they can be used to determine specificity, kinetics, affinity, and concentration with respect to the interactions between two or more molecules, where one of the molecules is attached to a solid sensing surface. Reaction kinetics correspond to both an association and a dissociation rate at which an analyte interacts with the bound detection molecule. Affinity refers to the strength with which an analyte binds to the detecting molecule. Specificity refers to the propensity of a molecule to bind to the detecting molecule to the exclusion of other molecules. SPR spectra have been used in studies involving many types of molecules including proteins, peptides, nucleic acids, carbohydrates, lipids, and low molecular weight substances (e.g., hormones and pharmaceuticals).
One analytical technique, known as SPR based bio-sensing, has been developed to enable direct measurements of the association of ligands with receptors, without the use of indirect labels, such as fluorescent markers and radioactive molecular tags. This label free direct sensing technique reduces the time and workload required to perform assays, and minimizes the risk of producing misleading results caused by molecular changes induced by the use of indirect labels. Another important aspect of the bio-sensing technique is that SPR based bio-sensing enables bio-molecular interactions to be measured continuously and in real-time, thereby enabling the determination of association and dissociation kinetic data in contrast to traditional “end point” analytical methods.
The utility and acceptance of SPR based bio-sensing is evident from the over 2,500 peer-reviewed scientific papers that have been published, which cite the use of SPR technology. To date, there is an estimated installed base of 1,500 research grade SPR analytical instruments in basic and applied research laboratories at universities, national research centers, and major pharmaceutical and biotechnology companies around the world. The diversity of recently published articles relating to bio-molecular interaction analysis include such applications as drug discovery (lead identification and target validation), ligand fishing, comparative binding specificity, mutation studies, cell signaling, multi-molecular complexes, immune regulation, immunoassays, vaccine development, and chromatographic development. Such SPR based research tools are of great value to researchers involved in basic and applied life sciences who are studying the function of molecules in biological systems.
Over the past decade, interest in the unique optical properties of metallic and semiconductor nanoparticles has increased considerably with respect to the use of suspensions and films incorporating these nanoparticles for the purposes of exciting surface plasmons to enable the detection of SPR spectra. In addition, surface enhanced Raman spectroscopy for infrared absorbance spectral information and surface enhanced fluorescence for enhanced fluorescence stimulation can also be detected. Nanoparticles are particles that are less than 100 nanometers in diameter. They display large absorbance bands in the visible wavelength spectrum yielding colorful colloidal suspensions. The physical origin of the light absorbance is due to incident light energy coupling to a coherent oscillation of the conduction band electrons on the metallic nanoparticle. This coupling of incident light is unique to discrete nanoparticles and films formed of nanoparticles (referred to as metallic island films). Achieving SPR with ordinary bulk materials requires the use of a prism, grating, or optical fiber to increase the horizontal component of the incident light wave vector (i.e., to achieve the required coupling).
Historically, gold nanoparticles have been used as a pigment in stained glass as early as 350 years ago. Chemist and physicist Michael Faraday first recognized that the color of this stained glass was a result of the metallic gold being in a colloidal form, and Gustaf Mie explained this phenomenon theoretically in 1908, by solving Maxwell's equation for absorption and scattering of electromagnetic radiation by a spherical particle.
Recently, sensor devices have been developed in the known art to exploit the unique optical properties of these nanoparticles. SPR measurements have been made using gold nanoparticle suspensions to detect biomolecular interactions in real time by monitoring the absorbance of colloidal suspensions. Similarly, SPR has been excited using polystyrene and silica beads with silver and gold island films and hollow gold nanoshells. However, to date, all such measurements have been performed primarily on bulk homogeneous suspensions of nanoparticles, due to the challenge of individually addressing and detecting these small objects.
For example, FIGS. 1A and 1B schematically illustrate a simplified version of a prior art SPR detection device including a single channel SPR bulk optic prism based sensor 10, which includes a prism 12. The base of prism 12 is covered with a layer 14 of gold about 550 Ängstroms thick. The gold film is pre-functionalized with a defined detecting molecule 16, such as an antigen. A biological fluid sample containing a corresponding analyte 32 (such as an antibody) is brought into contact with gold layer 14 and detecting molecules 16 by introducing the sample fluid into a flow cell 22 (note gold layer 14 and detecting molecules 16 together represent a sensor surface 20, which is in fluid communication with flow cell 22). A range of angles of monochromatic light 24 are directed towards and reflected from sensor surface 20. SPR arises through the coupling of energy between the incident photons of light with free electron oscillations (“plasmons”) occurring at a gold film/liquid chemical sample interface at sensor surface 20. This interaction can cause a reduction in an intensity of reflected light 28 for a given angle 30, resulting in an absorbance or “resonance” dip 31 in a measured reflectance spectrum 34 (see FIG. 1C). This resonance can also be observed in the wavelength domain if white light is introduced at an optimal fixed angle of incidence. When an analyte 32 binds to immobilized detecting molecule 16 on sensor surface 20 (see analyte 32a and molecule 16a of FIG. 1B), the local mass concentration of molecules changes, which causes a corresponding change in the local refractive index close to sensor surface 20. The resultant increase in the refractive index causes a shift in the resonance angle, from angle 30 as illustrated in FIG. 1A, to an angle 33 in FIG. 1B. Angle 33 results in a “resonance” dip 35 in a measured reflectance spectrum 37, which is readily distinguishable from spectrum 34 (no binding event, so the reflectance angle is unchanged). Sensor 10 enables data collection to be performed continuously and in real-time, and some systems enable the user to observe the binding events in real time on a personal computer.
This bio-sensing technique was first reported in 1983, and first commercialized in 1990. Since then many different optical geometries have been explored including: (i) the Otto configuration, which utilizes an air gap between the optical coupling prism and the SPR supporting metal; (ii) the Kretschmann configuration, which eliminates the need for an air gap in favor of the metal film directly deposited upon the prism base; (iii) the use of a diffraction grating to excite SPR; (iv) an optical fiber configuration, wherein metal is deposited cylindrically around the fiber core; (v) planar/channel waveguide configurations with retro-reflective elements; (vi) microstructure systems that have an integrated light source, detector, and guiding optics, including a capillary configuration in which SPR is excited in the interior capillary walls; (vi) use of gold island films; and (vii) two-dimensional (2D) imaging techniques for SPR array-based sensing.
FIG. 1D graphically illustrates a typical SPR response curve 38 based on the association and dissociation of two bio-molecules. Curve 38 can be separated into four well-defined segments, each relating to a specific portion of an association/disassociation cycle. The portion of the cycle corresponding to segment A is schematically illustrated in FIG. 1E. Flow cell 22 is in fluid communication with detecting molecules 16, which are bound to gold layer 14. While no prism is shown in conjunction with FIGS. 1E–1H, it should be understood that the flow cells of FIGS. 1E–1H are used in with a prism, as shown in sensor 10 of FIGS. 1A and 1B.
Referring once again to FIG. 1E, since analyte molecules are currently not present in flow cell 22, there is no change in the angle of incidence in section A of spectrum 38 (FIG. 1D). Generally the flow cell is filled with a buffer solution during this time period. Portion A of spectrum 38 is thus referred to as a baseline response.
In FIG. 1F (corresponding to portion B of spectrum 38), molecules of analyte 32 are introduced into flow cell 22 (i.e., a sample fluid containing the analyte is introduced into the flow cell). Some of the molecules of analyte 32 bind to detecting molecules 16, and the angle of incidence changes over time. Response curve 38 of FIG. 1D typically represents a time period of about 5 to 20 minutes in duration. This response level indicates the baseline response. During this “association” period, the analyte binds to the surface, thereby increasing the refractive index, causing the SPR resonant angle to increase (note the rise in portion B of spectrum 38).
In FIG. 1G (corresponding to portion C of response curve 38), no additional analyte is introduced into the flow cell. Instead, the flow cell is flushed with a buffer solution. This step results in analytes being released from detection molecules 16, as the bound analytes 32 attempt to reach an equilibrium with the buffer solution. The decrease in the amount of bound analyte is reflected in a dip in spectrum 38.
In FIG. 1G (corresponding to portion D of spectrum 38), the flow cell is flushed with an acidic solution, which ensures that any residual bound analytes are removed from the detection molecules, thereby regenerating the sensor surface. This “regeneration” step enables the sensor surface to be returned to its original baseline configuration, so that further analyses can be performed. As noted above, the data collected during portions A-D of spectrum 38 (often referred to as a Sensorgram) enable the user to determine kinetics, concentration, binding specificity, and affinity.
FIG. 2 shows a different prior art technique that has been developed, which also involves exciting and detecting SPR on gold and silver nanoparticles. Chemical and biological sensing applications using nanoparticles have been performed primarily by measuring a transmitted light intensity 80 through a high concentration of suspended particles 82. The resultant spectrum 84 has an absorbance dip 86 due to the excitation of SPR at a certain coupling wavelength 88, as shown in FIG. 2. The exact position and shape of the SPR spectrum is a function of such factors as the metal used, the bulk solution and adsorbed film complex refractive indices, the adsorbed film thickness, the nanoparticle morphology (size and shape), and inter-particle coupling effects (e.g. the concentration and proximity of nanoparticles to one another). For small nanoparticles compared to the wavelength, λ, the extinction cross section for the nanoparticles, can be approximated as indicated in Eq. 1, as follows:
                              σ          ext                ≈                                            9              ⁢              V              ⁢                                                          ⁢                              ɛ                b                                  3                  /                  2                                                      c                    ·                                                    ωɛ                2                            ⁡                              (                ω                )                                                                                      [                                                                                    ɛ                        1                                            ⁡                                              (                        ω                        )                                                              +                                          2                      ⁢                                              ɛ                        b                                                                              ]                                2                            +                                                                    ɛ                    2                                    ⁡                                      (                                          ω                      2                                        )                                                  2                                                                        (        1        )            where V is the spherical nanoparticle volume, c is the speed of light, ω is the angular frequency of the incident light, εb is the permittivity of the surrounding bulk dielectric medium (assumed to be relatively independent of the frequency of light), ε1(ω)) and ε2(ω) denote the real and imaginary parts of the metal permittivity, or more specifically, (ε(ω)=ε1(ω)+iε2(ω)).
For nanoparticle SPR measurements, the maximum absorbance wavelength, λspr (SPR coupling wavelength) dependence on refractive index is not as sensitive as the bulk thin film SPR measurements. Sensitivity of a 75 nanometer shift in the SPR coupling wavelength per refractive index unit (RIU) is reported, as compared to 3000 nanometer shift per RIU for bulk film SPR devices. Thus, gold nanoparticle SPR measurement based methods are 40 times less sensitive. However, additional geometries, including gold/silver alloy nanoparticles, ellipsoidal nanoparticles, triangular nanoparticles, and hollow nanoshells have been reported as having increased sensitivities up to six fold (400 nm wavelength shift per RIU).
Although bulk SPR devices exhibit increased sensitivity to refractive index over SPR nanoparticle devices, the nanoparticles have an advantage with respect to the sensitivity of adsorption of molecules to the gold surface. Specifically, the decay length of the electric field extending from the gold/chemical sample interface is approximately 20 times shorter for that of nanoparticle colloidal gold versus bulk thin gold film. Therefore, because nanoparticles have more energy confined closer to the gold surface, these particles are more surface sensitive and will yield a larger signal during receptor/ligand interactions.
However, the above mentioned prior art techniques are currently limited by throughput, mass transport diffusion, and depletion of small concentrations of analytes. Commercial SPR biosensors are currently limited to four-channel detection. This fact, and the relatively high degree of training necessary to operate these instruments and analyze the results, currently limit SPR analytical use in the laboratory. In contrast, other bio-molecular analytical methods, such as immunological assays, and spectroscopic techniques (absorption, fluorescence spectroscopy, and fluorescent polarization) have kept up with increased analytical demands by making available instruments having, for example, 96, 384, and 1,536 micro-wells. It should be noted that there are several publications directed towards multi-spot or 2D array SPR sensors. However, most if not all of these approaches are directed toward optical configurations that can only detect a single angle or single wavelength intensity. Therefore, changes in the association or dissociation of bio-molecules are detected as an intensity change, which has limited sensitivity and limited dynamic range compared to full spectral SPR data, where the entire angular or wavelength spectrum is measured, enabling a high precision measurement of the coupling angle or wavelength.
Current commercial SPR instrumentation uses a fixed sensor having a gold layer capable of supporting SPR, such as the traditional SPR bulk optic prism based sensor shown in FIGS. 1A and 1B. The bio-molecular receptor molecules attached to the gold layer are analyte specific. The analyte is brought to this sensor surface via fluidics, and the analyte associates with the bound receptor molecules on the sensor surface. Current planar embodiments are severely mass transport limited by diffusion to time scales on the order of 16 to 160 minutes for analytes at bulk concentrations less than 10−7 M.
Finally, current commercial SPR instrumentation uses sensors that have relatively large areas (e.g., four, twelve, and a hundred square millimeters). Because the SPR signal is proportional to the density of binding, having a large sensor area limits the analyte sensitivity, since low concentration analyte binding serves to deplete the analyte concentration near the surface.
It would therefore be desirable to provide apparatus and a method that address and detect individual nanoparticles and particles, enabling high throughput and full spectrum SPR measurement, measuring the association of molecules free in solution via SPR emitted from nanoparticles and micro particles suspended in solution, employing a significantly reduced sensor area with improved analyte sensitivity, providing a label-free direct sensing approach that reduces time and workload needed to carryout assays, and measuring biomolecular interactions continuously and in real-time. The prior art does not teach or suggest a complete solution to the problems discussed above.